JITV Vol. 20 No 2 Th. 2015: 148-157

Curcumin Effect on Bleomycin-Induced Pulmonary Fibrosis in Mus musculus

Rahmi A1, Setiyono A2, Juniantito V2

1Graduate School of Animal Biomedical Science, Faculty of Veterinary Medicine, Bogor Agricultural University 2Division of Pathology, Department of Clinic, Reproduction and Pathology, Faculty of Veterinary Medicine, Bogor Agricultural University E-mail: [email protected]; [email protected]

(received 31-03-2015; revised 29-05-2015; accepted 04-06-2015)

ABSTRAK

Rahmi A, Setiyono A, Juniantito V. 2015. Pengaruh kurkumin terhadap fibrosis paru-paru akibat aplikasi bleomisin pada Mus musculus. JITV 20(2): 148-157. DOI: http://dx.doi.org/10.14334/jitv.v20i2.1169 Kurkumin merupakan bahan aktif utama dari tanaman kunyit (Curcuma longa) diketahui memiliki aktivitas sebagai anti- oksidan dan anti-inflamasi. Bleomisin merupakan salah satu obat anti-kanker yang dapat menginduksi fibrosis paru-paru pada manusia dan hewan. Tujuan penelitian ini adalah untuk mengetahui efek biologis kurkumin pada fibrosis paru-paru yang diinduksi bleomisin pada mencit. Sebanyak 16 ekor mencit galur ddy dibagi dalam 4 kelompok perlakuan: (i) kontrol, 100 µl aquadest steril diinjeksikan secara SC, (ii) bleomisin (BLM), 100 µl bleomisin konsentrasi 1 mg/ml diinjeksikan secara SC, (iii) kurkumin (CMN), 100 µl aquadest steril diinjeksikan secara SC dan 100 mg/kg BB kurkumin dalam 0,5% carboxymethylcellulose (CMC) yang diinjeksikan secara IP, dan (iv) BLM+CMN, 100 µl bleomisin dengan konsentrasi 1 mg/ml diinjeksikan secara SC dan 100 mg/kg BB kurkumin dalam 0,5% CMC diinjeksikan secara IP. Semua perlakuan diberikan setiap hari selama 4 minggu. Organ paru-paru dikoleksi dalam 10% buffered neutral formalin (BNF). Pengamatan histopatogi dengan pewarnaan hematoksilin-eosin (HE) dan Masson’s trichrome (MT) untuk diamati tebal dinding alveol dan luas daerah jaringan ikat. Hasil penelitian menunjukkan bahwa pada kelompok perlakuan bleomisin (BLM) terjadi peningkatan luas jaringan ikat dan tebal dinding alveol secara signifikan jika dibandingkan dengan kontrol. Sementara itu, pemberian kurkumin pada mencit yang mendapatkan induksi bleomisin (kelompok BLM+CMN), menyebabkan terjadinya penurunan signifikan luas jaringan ikat dan tebal dinding alveol. Dapat disimpulkan kurkumin memiliki aktivitas sebagai inhibitor fibrogenesis untuk mengurangi keparahan fibrosis paru-paru akibat aplikasi bleomisin pada mencit. Kata Kunci: Bleomisin, Fibrosis Paru-paru, Mencit, Kurkumin

ABSTRACT

Rahmi A, Setiyono A, Juniantito V. 2015. Curcumin effect on Bleomycin-induced pulmonary fibrosis in Mus musculus. JITV 20(2): 148-157. DOI: http://dx.doi.org/10.14334/jitv.v20i2.1169 Curcumin, a curcuminoid compound of turmeric has been demonstrated to have anti-oxidant and anti-inflammatory properties. Bleomycin (BLM) is an anti-cancer drug induced pulmonary fibrosis in human and animals. This study was aimed to investigate biological effects of curcumin on bleomycin-induced pulmonary fibrosis in mice (Mus musculus) through pathomorphological assessment. In this study, 16 mice ddy strain were divided into four groups, namely (i) control, mice were subcutaneously (SC) injected with 100 µl sterilized aquadest in dorsal skin, (ii) BLM group, injected SC with 100 µl of 1 mg/ml BLM in dorsal skin, (iii) Curcumin (CMN) group, mice were intraperitoneally (IP) injected with 100 mg/kg body weight (BW) curcumin dissolved in 0,5% carboxy methyl cellulose (CMC) and injected with 100 µl sterilized aquadest SC, (iv) BLM+CMN group, injected SC with 100 µl of BLM 1 mg/ml and injected IP with 100 mg/kg BW CMN in 0,5% CMC. All treatments were performed daily for four weeks period. The lung samples were collected and fixed in buffered neutral formalin (BNF) 10%. Histopathological evaluation was performed with hematoxylin-eosin (HE) and Masson’s trichrome (MT) stains. The results showed that BLM treatment significantly increased fibrosis area and alveolar wall area fraction as compared to control. OIn the other hand, CMN treatment significantly reduced fibrosis area and alveolar wall area fraction in mice treated with BLM. In conclusion, our study showed that CMN treatment may inhibit lung fibrogenesis in BLM-induced pulmonary fibrosis. Key Words: Bleomycin, Curcumin, Mice, Pulmonary Fibrosis

INTRODUCTION and radiographic findings (Todd et al. 2012). Many factors cause this pulmonary fibrosis, such as it is Pulmonary fibrosis is a chronic pulmonary disease exposured by air poison, particular pulmonary disease, characterized by pathological lesions in the form of effect of radiation therapy and chemotherapy (Ley & extracellular matrix and tissues component changes, Collard 2013). followed by clinical symptoms, physiological disorders,

148 Rahmi et al. Curcumin effect on Bleomycin-induced pulmonary fibrosis in Mus musculus

Bleomycin (BLM) is used as a chemotherapy agent synthesis and inhibiting free radicals releasing in the for various cancers. This antibiotic of antitumor is a inflammatory cells (Jurenka 2009; Marçal et al. 2012). derivative glycopeptide isolated from Streptomyces Some studies have been conducted to determine verticillus (Yamamoto 2010). BLM has strong curcumin effect as anti-inflammation and its potential to antitumor activity. Clinically, BLM is used as tumor prevent the fibrosis (Jurenka 2009; Beevers & Huang therapy such as squamous cell carcinoma at around 2011; Kardena & Winaya 2011). Curcumin can head and neck (including mouth, tongue, tonsils, decrease fibrosis level in liver, kidney, and lung of nasopharynx, oropharynx, sinus, palate, lips, buccal laboratory animal (Beevers & Huang 2011). Therefore, mucosa, the gums, the epiglottis, and larynx), malignant curcumin can be used as potential candidate of anti- lymphoma, testicular carcinoma, and malignant pleural fibrosis preparate, especially in pulmonary fibrosis. effusion (Chu et al. 2010). This study was aimed to see the curcumin potential in The main limitation of BLM therapy is its side preventing the side effect of BLM use in cancer patients effect which causes alveolitis fibrosis (Matsushita et al. who are undergoing a chemotherapy. 2008). Frusch et al. (2012) said that there are several syndromes in the lung linked with BLM utilization, that is bronchiolitis obliterans, hypersensitivity eosinophils, MATERIALS AND METHODS and interstitial pneumonitis which will thrive become pulmonary fibrosis. Besides, BLM-induced pneumonitis Time and place can reach 46% in patients who receive BLM treatment. Mortality level of the pulmonary fibrosis disease is This study was conducted during December 2014 - around 10-20% with 2-3% from patients treated by March 2015 in Laboratory Animals Management Unit BLM. Brugge et al. (2013) said that BLM therapy side (UPHL), Faculty of Veterinary Medicine, Bogor effect could cause pneumonitis. Agricultural University and Histopathology Laboratory, Fibrosis mechanism in lung due to the side effect of Pathology Division, Faculty of Veterinary Medicine, BLM utilization has not known. Some factors that have Bogor Agricultural University. role in pulmonary fibrosis in BLM therapy are oxidative stress, BLM- deactivation, genetic, Inducer material of pulmonary fibrosis and the release of inflammatory cytokines (Brugge et al. 2013; Reinert et al. 2013). BLM use in human also Bleocin (Bleomycin hydrochloride 15 mg, Kalbe causes other side effect, such as an abnormality skin Farma, Jakarta, Indonesia) was diluted in 15 ml sterile characterizes the scleroderma (Juniantito et al. 2013). aquadest to reach concentration of 1 mg/ml. As much as Fibrosis pathogenesis (fibrogenesis) is divided in 100 µL of the solution was injected subcutaneously several overlapping phases, namely inflammation, (SC) on the back skin for BLM and BLM+CMN group connective tissue proliferation, and intractable fibrosis everyday for 4 weeks. (Matsushita et al. 2008; Reinert et al. 2013). Retardation in one step of fibrogenesis causes a Curcumin decreasing of connective tissue formation (Loomis- King et al. 2013). BLM could stimulate endothelial Curcumin active ingredients (Biopurify, Chengdu, cells, macrophages, and fibroblasts to induce synthesis China) was diluted in 0.5% carboxymethylcellulose of inflammatory mediators especially proinflammatory (CMC) and injected 100 mg/kg of body weight by and fibrogenic cytokines, inducing apoptosis, and free intraperitoneal injection (IP) in CMN and BLM+CMN radicals synthesis (Yamamoto 2010; Yamamoto & group everyday for 4 weeks. Katayana 2011). It was thought that prevention of fibrosis might be mediated by inhibition of Experimental procedure inflammation with anti-inflammatory substances (Basnet & Skalko-Basnet 2011; Kardena & Winaya All of procedures conducted in this study met the 2011). requirement of Animal Ethics Commission of Bogor Curcumin (diferuloylmethane) is an active Agricultural University Number 25-2014 IPB. Sixteen 4 ingredient in turmeric, in addition, it is also known as weeks old male ddy strain mice with body weight an antioxidant (Zhang et al. 2011). Turmeric is widely around 20-25 gram (The National Agency of Food and available plant in Indonesia which is used often as spice Drug Control (NA-DFC), Jakarta, Indonesia) were used and herbal ingredient. Anti inflammation effect of and divided into 4 groups. There were 4 mice in each curcumin is likely because of a high pleiotropic group. molecule which able to interact with and related to the This study was divided into 4 treatment groups, most transcription factors in inflammatory mediators namely: (i) control, injected subcutaneously by 100 µL sterile aquadest in the back skin, (ii) BLM, injected

149 JITV Vol. 20 No 2 Th. 2015: 148-157 subcutaneously in the back skin with 100 µL BLM of 1 customized for contrast, brightness, and threshold color. mg/mL concentration, (iii) CMN, injected Image analysis program detected the width of blue area subcutaneously with 100 µL sterile aquadest in the back which indicates collagen area or connective tissue in skin and injected intraperitoneally with curcumin of 100 each visual field and presented in the form of mg/kg of body weight in 0.5% CMC, and (iv) percentage. BLM+CMN, injected subcutaneously with 100 µL BLM of1 mg/mL concentration and injected Alveolar wall thickness intraperitoneally with curcumin of 100 mg/kg of body weight in 0.5% CMC. Injection of the curcumin by Alveoli wall thickness quantitatively was counted intraperitoneal injection was adapted from Li et al. by analyzing tissue slide which has been stained by HE. (2013). As much as 20 visual fields per treatment group were This study was done in three steps, namely: (i) randomly selected using video camera showed on the acclimatization, the experimental animals were adapted colored screen. The wide of each visual field was in new cage for 2 weeks, (ii) treatments, the 326.40×184.00 µm2. Alveoli wall thick measurement experimental animals were treated in accordance to the was done 10 times of every visual field, and the data respective groups everyday for 4 weeks, (iii) presented in the form of average. termination, the experimental animal were euthanized by giving Ketamine HCL of 0.2 mL/head (AVMA Data analysis 2013). Furthermore, left lobus of lung was collected to be made histopathology preparates, then stained by Percentage of width of connective tissue and alveoli hematoxylin-eosin (HE) for observation of tissue wall thickness were analyzed by SAS 1.9 software for structure of the lung (Fischer et al. 2006) and MicrosoftWindows ANOVA and presented in form of Masson’strichrome (MT) to see the presence of average and standard deviation. Further, Duncan connective tissue (Suvik & Effendy 2012). advance test was done to determine whether there is a significant differences between the treatments or not. Histopathological assesment by HE and MT stains

Lung of BNF 10% was sliced in 3 mm and inserted RESULT AND DISCUSSION into tissue cassette for dehydration process, clearing and paraffin infiltration using automatic tissue Histology Result by Hematoxylin-Eosin Staining processor. Chunk organs, further was printed in the paraffin until paraffin block formed. For histology test, Figure 1 is photomicrography of lung tissue of each the paraffin block was sliced by rotary microtome in 3- treatment. Figure 1A is a photomicrography of control 5 μm of thickness. Cutting results were placed in object group of lung tissue. In this figure, normal alveoli wall glass to be deparafinized and rehydrated for tissue structure is 3.7±0.6 μm. The same thing occurred in staining by hematoxylin-eosin and Masson’s trichrome. CMN group (Figure 1C). It is shown that lung with All of the cutting results were observed by a light normal alveoli wall structure of 3.4±0.5 μm. microscope which was connected to the computer. Figur 1B is a photomicrography of BLM group. In Observed parameters of this organ were connective this group, it is shown an inflammation accompanied by tissue width and alveoli wall thickness. an increase in cell number in interstitium, therefore alveoli wall become thicker (24.9±6.3 μm) than the Connective tissue width control group. Besides, there was an infiltration of inflammatory cells dominated by macrophages and Connective tissue width was quantitatively counted lymphocytes. Alveoli wall thickening resulted in width by Image J software (http://imagej.nih.gov//ij/; NIH, of alveoli area became smaller, therefore the air entered Maryland, USA), by analyzing tissue slide which has the lung became limited. Rienert et al. (2013) said that stained by modification of MT stains (Suvik & Effendy thickening of the alveoli wall would cause difficulty in 2012). Image J software utilization is to decrease the breathing (anoxia). In the BLM+CMN group (Figure level of counting subjectivity by scoring method. By 1D), it is shown lesion similar to the BLM group, that is using 40x objective lens magnification, as much as 20 the presence of inflammation, increasing in cell number visual fields per treatment group randomly selected by in interstitial and the thickening of the alveoli wall video camera (Indomikro HDMI camera) which was (9.9±2.6 μm) with lower severity compared to the BLM shown on colored screen. The width of each visual field group. was 326.40×184.00 µm2. Furthermore, figure was

150 Rahmi et al. Curcumin effect on Bleomycin-induced pulmonary fibrosis in Mus musculus

Figure 1. Photomicrography of lung tissues by HE staining of each treatment group: Control (A); BLM (B); CMN (C); and BLM+CMN (D)

Figure 2. Photomicrography of lung tissues by MT staining of each treatment group: Control (A); BLM (B); CMN (C); BLM+CMN (D)

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Yamamoto & Nishioka (2000) said that in mice Width of connective tissue everyday induced by BLM SC for 4 weeks, there was pulmonary fibrosis characterized by mononuclear cell Measurement of connective tissue width in the lung infiltration, ederma, and septum alveolar thickening due was done to determine extent of lung tissue damage due to decrease in number of macrophages alveolar and to BLM application. In figure 3, it is not seen the lymphocyte. Besides, Izbicki et al. (2002) found that presence of a significant differences of the connective there were perivascular and lymphocytes peribronkial tissue between control group (2.4±0.8%) and CMN (day 3), subcapsular fibrosis (day 6), and decreasing in group (1.5±0.6%). However, in BLM group number of macrophages alveolar and septum alveolar (15.0±2.4%), there was significantly wider connective (day 14) in mice’s lung histology induced by pulmonary tissue value (P<0.05) compared to control group. In the fibrosis with single dose BLM by intratracheal. BLM+CMN group (6.2±2.0%), there was significantly lesser width connective tissue (P<0.05) compared to the Histology result by Masson’s Trichrome (MT) BLM group. This research result was in line with staining Izbicki et al. (2002) that there was an increase in connective tissue width of mice lung induced by single Tissue staining techniques by MT is addressed to dose Bleomycin by intratracheal. Besides, Liu et al. identify the presence of the connective tissue or (2013) also reported that there were an inflammation collagen in the tissue. Result of the lung tissue staining and fibrosis in interstitial of mice lung tissue induced by by MT technique was presented in Figure 2. BLM through intratracheal. Figure 2A (control) and 2C (CMN) shows normal Wide development of connective tissue in BLM structure of lung tissues with thin alveoli wall (3.7±0.6 group was caused by BLM activity which may induce μm). In the two figures do not show an excessive blue pulmonary fibrosis by causing inflammation, than it structure, indicating that in the two group there is no developed become proliferation of fibroblast. Fibroblast excessive collagen accumulation. On the contrary, is a cell which produced extracellular matrix (Todd et figure 2B (BLM) shows blue accumulation which al. 2012). Yamamoto & Katayama (2011) said that extends almost to the entire visual field. The blue color BLM can stimulate endothelia cell, macrophage, and on lung slide colored by MT indicating the presence of fibroblast to induce inflammatory mediator. The accumulation connective tissue or collagen (Suvik & inflammatory mediator will induce proinflammatory Effendy 2013). Besides, in the BLM group was also cytokines, cytokine fibrogenic, transforming growth seen smaller alveoli diameter compared to the control factor-β (TGF-β), apoptosis, and the release of free group. Figure 2D (BLM+CMN) shows light radicals. accumulation of collagen visualized by blue color and looked like thickening of the alveoli wall.

15.0±2.4b

6.2±2.0c

2.4±0.8a 1.5±0.4a

Wide of tissue (%) connective

Control BLM CMN BLM+CMN

(group of treatment)

Figure 3. The wide of connective tissue of each treatment group, different superscript word shows significant difference (P<0.05)

152 Rahmi et al. Curcumin effect on Bleomycin-induced pulmonary fibrosis in Mus musculus

Reinert et al. (2013) reported that low BLM- connective tissue compared to the BLM group (Figure hydrolase enzyme activity can be a predisposing factor 3). In line with Punithavathi et al. (2000) that there was of pulmonary fibrosis due to the BLM application. a decreasing of alveolar macrophage amount, Through in vivo, the BLM will be metabolized become superoxide and nitrikoksida in rats given oral curcumin non-toxic molecule with Bleomycin-hydrolase enzyme treatment after single dose Bleomycin induction by help (Haston et al. 2002). This enzyme is cytosolic intratracheal. So, in his research, it was concluded that aminopeptidase group which has low activity in lung curcumin was a potential material as an anti- and skin (Reinert et al. 2013). Therefore, BLM inflammatory and anti-fibrogenic in BLM-induced application may cause the pulmonary fibrosis. The pulmonary fibrosis. fibrosis in the lung is started by an inflammatory. The Bleomycin as an antibiotic antitumor agent, widely inflammatory will develop into proliferation of used for livestocks, such as pig (Balazs et al. 1994) and fibroblasts into myofibroblast (cell produces collagen), sheep (Organ et al. 2015). Same as in laboratory and it finished by persistent formation of connective animals, BLM utilization in pig and sheep also caused tissue. Mechanism of fibrosis induction by BLM was side effect such as an inflammation into pulmonary caused by increasing secretion of extracellular matrix fibrosis (Balazs et al. 1994; Organ et al. 2015). proteins because of cytokine fibrogenic effect which Pathogenesis mechanism of pulmonary fibrosis due to was released by macrophages, endothelial cells, and BLM application in livestocks was alike with the fibroblasts (Reinert et al. 2013). Besides, BLM in the laboratory animals, so that was possible to be given cells will bind with iron ion (Fe+2) and oxygen forming curcumin as anti-inflammation and antioxidant to Fe+3 ion which produce the free radicals, so that cause reduce fibrogenesis severity in the pulmonary fibrosis DNA damage, RNA degradation, and inducing the cases due to BLM application. apoptosis (Yamamoto 2010). Moreover, the free The decreasing of width of lung connective tissue in radicals or reactive oxygen species can produce direct this study may be caused by 2 factors. First, the toxicity through participation in redox reactions and curcumin can directly inactivate the bleocymin activity, subsequent fatty acid oxidation, which leads to so that inflammation did not occur. Second, activity of membrane instability. Oxidant can cause inflammatory anti-inflammation from the curcumin causes the reaction within be lung. The inflammatory mediators inflammation become lower, so that fibrosis can be like cytokines such as interleukin-1, macrophage pressed. The second one is closer with pathogenesis of inflammatory protein-1, platelet-derived growth factor, fibrosis inhibition by the curcumin. This was caused by and TGF-β are released from alveolar macrophages in BLM induction and curcumin therapy conducted animal models of bleomycin toxicity, resulting in through 2 different ways. Bleomycin induction was fibrosis. Damage and activation of alveolar epithelial done subcutaneously, whereas curcumin therapy was cells may result in the release of cytokines and growth done by intraperitoneal. Both of subcutaneous and factors that slimulate proliferation of myofibroblast and intraperitoneal ways will interact systematically in the secretion of pathologic extracellular matrix, leading to body, so that increasing width of lung connective tissue fibrosis. due to Bleomycin application possibly hampered by The inflammation due to BLM application will anti-inflammation activity from the curcumin. Besides, cause body to response to repair the tissue by involving the curcumin was known to have effectiveness as an connective tissue reconstitution from the fibroblast into inhibitor receptor TGF-β, so that TGF-β production can myofibroblast (Reinert et al. 2012). Myofibroblast is the be inhibited (Mo et al. 2012). main component in fibrosis process or wound healing. Prasad et al. (2014) said intraperitonel injection of This myofibriblast will produce extracellular matrix, so curcumin more often applied to animals than to human. it will cause extracellular matrix proteins accumulation. Besides, the bioavailability compound of curcumin via Previous study conducted by Xu et al. (2009) showed intraperitoneal injection is higher than gavage. that TGF-β is cytokine inducer of differentiation of Interestingly, oral curcumin treatment showed no effect fibroblast cell into myofibroblast cell. Yamamoto & on important measure of BLM-induced injury in mice, Nishioka (2004) presented that several actions of TGF- whereas intraperitoneal curcumin administration β are, among other as chemotactic macrophages and effectively inhibited inflammation and collagen fibroblasts, stimulating fibroblast proliferation, deposition along with a trend toward improved survival increasing extracellular matrix synthesis, decreasing of animal, and also reduced fibrotic progression even extracellular matrix proteinase regulation, and when administered after the acute bleomycin-induced increasing proteinase inhibitor regulation. So that inflammation had subsided (Smith et al. 2010). exacerbates the development of fibrotic lesions in the Smith et al (2010) said that curcumin administration lung. could significantly prevent lung inflammation and The opposite occurred in BLM+CMN group where collagen deposition in pulmonary fibrosis induced by there was a significant decrease (P<0.05) of width of BLM by intratracheal. Curcumin was high pleiotropic

153 JITV Vol. 20 No 2 Th. 2015: 148-157 molecule which able to interact and bind with the most radioprotective state prior to irradiation by inducing of inflammation molecule target (Jurenka 2009; Marçal protective expression as well as having potent et al. 2012). Curcumin has been reported having activity direct antioxidant scavenging activity. Li et al. (2013) as an anti-cancer (Wiken et al. 2011), anti-oxidant said curcumin application has a protective effect on the (Bhullar et al. 2013) and anti-inflammation (Basnet & acute hepatic injury induced by acetaminophen with Skalko-Basnet 2011). Turmeric bioactive compounds reduced acetaminophen-induced hepatocyte apoptosis had protective effect to heart cell which exposed to and that protection may be related to its inhibition of toxic chemicals such as tetrachloride (CCl4) (Prakash et lipid peroxidation and oxidative stress. al. 2008; Kardena & Winaya 2011). Yu et al. (2011) reported that curcumin administration can reduce Thickness of alveolar wall pancreatic tissue damage and another organs due to caerulein induction through inhibiting the release of Figure 4 shows that there is no significant difference inflammatory mediator of TNF-α cytokines. of alveolar wall between control group (3.7±0.6 μm) Curcumin has been reported to exhibit anti- and CMN group (3.4±0.5 μm). However, there was tumorigenic and chemo-preventive activities due to the significantly (P<0.05) higher alveolar wall thickness structural resemblance of dibenzoyilmethane (DBM) to value in BLM group (24.9±6.3 μm) compared to the the anti-inflammatory and aspirin-like skeleton of DBM control group. In BLM+CMN group (9.9±2.6 μm) there derivates (Lin et al. 2011). Villegas et al. (2011) was significantly (P<0.05) lower alveolar wall thickness reported curcumin has also demonstrated protective and value compared to BLM group. preventive effect in progression of colorectal colitis The results about width of connective tissue and cancer, which was collerated with a lowered immune- alveolar wall thickness in each treatment group were reactivity of beta-catenin and reduction of pro- presented in Table 1. inflammatory cytokine levels and a decrease of This research result is aligned with a research of inflammatory mediator’s overexpression. Izbicki et al. (2002) where alveolar wall thickening in Lee et al. (2010) evaluated that curcumin as a mice lung tissue induced by single dose BLM by potential dietary supplement in the setting of thoracic intratracheal. Alveolar wall thickening in BLM group radiotherapy in mice with creating a baseline

Figure 4. The Alveolar wall thickness in each treatment group, different superscript word shows significantly (P<0.05) difference

Table 1. Result of the wide of connective tissue and alveolar wall thickness Treatment Group Wide of connective tissue (%) Alveolar wall thickness (µm) Kontrol 2.39±0.78a 3.72±0.56a BLM 15.03±2.45b 24.93±6.26b CMN 1.50±0.61a 3.39±0.54a BLM+CMN 6.17±2.01c 9.95±2.60c

Different superscript in one same column shows significantly difference (P<0.05) n= 4

154 Rahmi et al. Curcumin effect on Bleomycin-induced pulmonary fibrosis in Mus musculus was caused by BLM administration continuously every CONCLUSION day, which eventually caused chronic type lesio forming. The BLM continuous administration caused Curcumin administration can inhibit fibrogenesis in formation of progressive pulmonary fibrosis lesion mice pulmonary fibrosis due to the BLM application. (Schwaiblmair et al. 2012; Reinert et al. 2013). Increase This information can be used as first information to wall thickness was caused by the existence of the prevent side effect of BLM utilization to cancer patients fibrosis, besides it can also caused by interstitial edema who has to undergo chemotherapy. (Izbicki et al. 2002). In this study, Bleomycin toxicity was caused by low activity of BLM-hydrolase enzyme in the lung causing ACKNOWLEDGEMENT the BLM not metabolized into intoxic molecule form. Reinert et al. (2013) said that low activity of BLM- This research was supported by lecturer and hydrolase enzyme in the lung resulted in an excessive pathology staffs, clinical department, reproduction, and apoptosis. Besides, the BLM can also stimulate pathology FKH-IPB. endothelial cell, macrophage, and fibroblast to induce inflammatory mediator, apoptosis, and free radicals REFERENCE (Yamamoto & Katayama 2011). The most common symptomps from bleomycin-induced hypersensitivity [AVMA] American Veterinary Medical Association. 2013. pneumonitis are exertional dispnea and non-productive AVMA guidelines for the euthanasia of animals: 2013 cough, with progressive pneumonitis dyspnea at rest, edition. Version 2013.0.1. [accessed 2/1/2015]. tachypnea, and cyanosis may occur (Sleijfer 2001). https://www.avma.org/KB/Policies/Documents/euthanas Bleomycin is one of xenobiotis which able to induce ia.pdf. radical oxygen synthesis excessively such as Balazs G, Noma S, Khan A, Eacobacci T, Herman PG. 1994. superoxide, hydrogen peroxide, peroxynitrite and Bleomycin-induced fibrosis in pig: evaluation with CT. hydroxyl radicals which is the mean mediator in Radiology. 191:269-272. http://dx.doi.org/10.1148/ inflammatory process in the lung (Oury et al. 2001). radiology.191.1.7510895. The inflammation will cause inflammatory cells Basnet P, Skalko-Basnet N. 2011. Curcumin: an anti- entering the tissue and releasing cytokine inflammation inflammatory molecule from a curry spice on the path to mediator (Reinert et al. 2013). Furthermore, the cancer treatment. Molecules. 16:4567-4598. cytokine caused fibroblast proliferation into http://dx.doi.org/10.3390/molecules16064567. myofibroblast. This inflammation and fibroblast Beevers CS, Huang S. 2011. Pharmacological and clinical proliferation will cause an increase of alveolar wall properties of curcumin. Botanics: Targets Therapy. thickness to the pulmonary fibrosis (Izbicki et al. 2002). 1:15-18. Curcumin administration significantly press alveolar wall thickness incidence. The curcumin can press Bhullar KS, Jha A, Youssef D, Rupasinghe HPV. 2013. apoptosis and inflammation incidence; this is related to Curcumin and its carbocyclic analogs: structure-activity in relation to antioxidant and selected biological its activity as anti-oxidant and anti-inflammation (Smith properties. Molecules. 18:5389-5404. http://dx.doi.org/ et al. 2010; Bhullar et al. 2013; Basnet & Skalko-Basnet 10.3390/molecules18055389. 2011). Smith et al. (2010) presented that curcumin administration significantly press pulmonary Brugge SS, Claasen HR, Dawson L. 2013. Drug induced lung inflammation and collagen deposition in the pulmonary injury- a case of fatal Bleomycin interstitial pneumonitis. Netherlands J Critical Care. 17:25-29. fibrosis induced by BLM. Punithavathi et al. (2000) said that the curcumin mediated the reduction of total Chu E, Harrold LJ, Tiedemann D, Copur MS. 2010. number of broncho-association limfoid follicles Chemotherapeutic and biologic drugs. In: Chu E, (BALF) cell by preventing inflammatory cell crossing DeVita VT, editors. Physicians’ cancer chemotheraphy endotel and epithelial basal membrane towards the drug manual. USA: Jones and Bartlett Publishers. p. 51- 54. inflammation location. This is because of the curcumin stabilized the endotel and epithelial basal membrane. Fischer AH, Jacobson KA, Rose J, Zeller R. 2006. Preparation Therefore, the curcumin activity as anti-oxidant and of cells and tissues for fluorescence microscopy. In: anti-inflammation can press alveolar wall thickness Spector DL, Goldman RD, editors. Basic methods in incidence in the lung due to BLM administration. microscopy. USA: Cold Spring Harbor Laboratory Press.

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